1. Field of the Invention
The invention relates generally to the field of plant biotechnology. More specifically, the invention relates to methods for selecting transformed plant cells using auxin-like herbicides as a selective agent.
2. Description of the Related Art
Transgenic crops are currently grown on more than 80.0 million hectares world-wide. Improved traits provided by transgenes have significantly increased productivity and in many instances decreased reliance on herbicides and insecticides that can potentially contaminate the environment. However, for transgenic crops to continue to be competitive in the market place, new value-added traits will be required.
In the production of transgenic plants, a particularly important step is the selection of transgenic cells. This is because only a small percentage of cells are typically transformed in any given transformation protocol. The use of a selectable marker gene allows those cells containing a marker gene to be selected away from those that do not. In attempts to stack multiple transgenes in a single plant, this can become particularly difficult, as multiple selectable marker genes are required. Additionally, while a number of selectable markers have previously been described, many do not confer a trait of any practical agronomic value and thus needlessly complicate regulatory approval. Alternatively, labor intensive steps must be taken to attempt to breed selectable markers out of a given transgenic plant. A selectable marker gene with dual functions of a selectable marker and a trait would thus be especially valuable.
Commonly used selectable marker genes for plant transformation are neomycin phosphotransferase II, isolated from Tn5 and conferring resistance to kanamycin (Fraley et al., 1983) and hygromycin phosphotransferase, which confers resistance to the antibiotic hygromycin (Vanden Elzen et al., 1985). Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, and the bleomycin resistance determinant (Hayford et al., 1988; Jones et al., 1987; Svab et al., 1990; Hille et al., 1986).
Other selectable marker genes for plant transformation not of bacterial origin are available. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase (Eichholtz et al., 1987; Shah et al., 1986; Charest et al., 1990). Among some herbicides that selectable marker genes confer resistance to are glyphosate, glufosinate, or bromoxynil (Comai et al., 1985; Gordon-Kamm et al., 1990; Stalker et al., 1988).
Genes encoding enzymes which inactivate herbicides and other xenophobic compounds have previously been isolated from a variety of prokaryotic and eukaryotic organisms. In some cases, these genes have been genetically engineered for successful expression in plants. Through this approach, plants have been developed which are tolerant to the herbicides 2,4-dichlorophenoxyacetic acid (Streber and Willmitzer, 1989), bromoxynil (Stalker et al., 1988), glyphosate (Comai et al., 1985) and phosphinothricin (De Block et al., 1987). While these plants have proven valuable in a commercial setting, plants tolerant to other herbicides are needed to avoid over reliance on any single herbicide and to increase options for managing difficult to control weed species.
In addition to the foregoing herbicides, there are auxin-like herbicides that mimic or act like natural plant growth regulators called auxins. Auxin-like herbicides appear to affect cell wall plasticity and nucleic acid metabolism, which can lead to uncontrolled cell division and growth. The injury symptoms caused by auxin-like herbicides includes epinastic bending and twisting of stems and petioles, leaf cupping and curling, and abnormal leaf shape and venation.
Dicamba is one example of an auxin-like herbicide and is used as a pre-emergent and post-emergent herbicide for the control of annual and perennial broadleaf weeds and several grassy weeds in corn, sorghum, small grains, pasture, hay, rangeland, sugarcane, asparagus, turf, and grass seed crops (Crop Protection Reference, 1995). Unfortunately, dicamba can injure many commercial crops and dicot plants such as soybeans, cotton, peas, potatoes, sunflowers, and canola are particularly sensitive to the herbicide. Despite this, auxin-like herbicides are very effective in controlling weed growth and thus are an important tool in agriculture. This is underscored by the development of weeds tolerant to other herbicides.
Recently, a gene for dicamba monooxygenase (DMO) was isolated from Pseudomonas maltophilia that confers tolerance to dicamba (US Patent Appln. 20030135879). DMO is involved in conversion of herbicidal dicamba (3,6-dichloro-o-anisic acid) to a non-toxic 3,6-dichlorosalicylic acid. This gene provides tolerance to dicamba in plants expressing the DMO gene. However, transformants containing the gene had to date only been selected using a separate selectable marker gene and techniques enabling use of a DMO gene as a direct selectable marker were not described. The need to use a separate selectable marker complicates the production of plants tolerant to auxin-like herbicides by requiring an additional gene on transformation vectors used and also presents regulatory hurdles.
Thus, there is a need in the art for new selectable marker genes and new herbicide tolerance genes. Particularly needed is a method for the selection of cells expressing a gene conferring tolerance to dicamba and other auxin-like herbicides that can be directly selected. A selectable marker gene with the dual function of a marker and a trait would eliminate the costs associated with preparing and tracking of two expression units during the development of a product and would facilitate the production of plants having valuable new traits.